Method of identifying cells expressing a protein having pre-B cell growth-supporting ability

ABSTRACT

A gene encoding a novel membrane protein polypeptide having pre-B cell growth-supporting ability, and a novel membrane protein polypeptide consisting of 180 amino acid residues having pre-B cell growth-supporting ability or a part of it. A method for producing said novel membrane protein polypeptide by preparing transformants by transforming a host cell with a vector containing said gene and culturing said transformants. A monoclonal antibody recognizing said novel membrane protein polypeptide. The above gene encodes the membrane protein having pre-B cell growth-supporting ability. The homogeneous and purified novel membrane protein polypeptide can be produced in large quantities and in addition, monoclonal antibodies recognizing said polypeptide can be produced; thus it becomes possible to identify rheumatoid arthritis (RA) and also prepare reagents for the clinical diagnosis thereof.

TECHNICAL FIELD

The present invention relates to a gene and a novel membrane proteinencoded by said gene, and more specifically, relates to a gene encodinga novel membrane protein polypeptide having pre-B cell growth-supportingability, a vector containing said gene, transformants transformed bysaid vector and a method for producing the novel membrane proteinpolypeptide by using said gene.

The present invention further relates to a monoclonal antibodyrecognizing a novel membrane protein polypeptide having pre-B cellgrowth-supporting ability.

The gene of the present invention encodes a novel membrane proteinpolypeptide enhancing pre-B cell growth-supporting ability on thesurface of synovial cells derived from patients with rheumatoidarthritis (RA). In the present invention, a homogeneous and purifiednovel membrane protein polypeptide having pre-B cell growth-supportingability can be produced in large quantities by transforming appropriatehost cells with a suitable vector in which the gene of the presentinvention is inserted. Thus, according to the present invention, itbecomes possible to identify rheumatoid arthritis (RA), and also preparereagents for the clinical diagnosis thereof.

BACKGROUND ART

Inflammatory cells in the synovial membrane and the synovial fluid ofpatients with rheumatoid arthritis (RA) are derived from peripheralblood and the migration of these cells to synovial membranes has notbeen explicated perfectly yet, but it is believed to be caused by acomplicated interaction between chemical signals given to cells andprotein (adhesion molecule) on cell membranes.

Various studies upon the significance of membrane proteins in arthritishave been performed. For example, it is known that an intercellularadhesion molecule-1 (hereinafter referred to as ICAM-1) is expressed onthe inner layer of the synovial membrane and the blood vessel of thesynovial membrane of patients with rheumatoid arthritis (RA), which is aligand of a T cell surface molecule LFA-1 and causes both adhesion andmigration of cells in the blood vessel wall [Hale et al.; ArthritisRheum., 32:22 (1989), and Haynes et al.; Springer Semin. Immunopathol.,11:163 (1989)].

Similarly, it is suggested that a vasocellular adhesion molecule-1(hereinafter referred to as VCAM-1), which is a ligand of integrin VLA-4expressed on T lymphoid cells (memory cells in particular) andmonocytes, is expressed on the synovial membrane and fibroblast-likesynovial cells of rheumatoid arthritis (RA) and osteoarthritis,[Morales-Ducret et al.; J. Immunol., 149:1424 (1992)], and further thata membrane protein called VAP-1 is expressed on the endothelial vein ofa synovial membrane and may work as a specific recognition structure ofleukocytes [Salmi et al.; Science, 257, 1407 (1992)].

The present inventors have engaged in extensive studies with a view toinvestigating the function of the bone marrow microenvironments indisorders causing abnormalities of B cells, and have found that thepre-B cell growth-supporting ability of bone marrow stromal cellsderived from patients with rheumatoid arthritis (RA) and multiplemyeloma (MM) is enhanced in comparison with that of healthydonor-derived bone marrow stromal cells and that the direct contact ofpre-B cells with stromal cells might play an essential role in thissupporting ability. And the present inventors have established novelstromal cell lines (RASV5-5, MMSV3-3) containing a molecule enhancingthe growth of pre-B cells by cell-lining stromal cells of patients, andhave found that the pre-B cell growth-supporting activity of thesestromal cell lines is most likely caused by unknown adhesion moleculesdifferent from known stem cell factors (SCF), ICAM, CD44, VCAM-1,LFA-1α, LFA-1β, NCAM and FLAM-1 [J. Immunol., 149:4088 (1992)]

Further, since it has been suggested that the synovial cell lineSynSV6-14 established from the synovial cell derived from patients withrheumatoid arthritis (RA) has pre-B cell growth-supporting abilitysimilarly to the stromal cell line RASV5-5 derived from the bone marrowof patients with rheumatoid arthritis (RA), the present inventors haveobtained a novel monoclonal antibody which responds to these cell linesbut does not respond to the stromal cell line NFSV1-1 derived from thehuman bone marrow having no pre-B cell growth-supporting ability, and atthe same time have succeeded in cloning genes encoding its antigenmembrane protein (Bst-1) (Japanese Patent Application No.5-141178/1993).

DISCLOSURE OF INVENTION

The present inventors have obtained a novel mouse monoclonal antibodyRS38 which responds to SynSV6-14 but does not respond to the healthybone marrow stromal cell line NFSV1-1 and recognizes a membrane proteindifferent from the above Bst-1 at the process of producing various mousemonoclonal antibodies recognizing a membrane protein expressed on thesynovial cell derived from patients with rheumatoid arthritis (RA) butnot expressed on the cell derived from healthy donors. Subsequently, thepresent inventors have succeeded in isolating clones encoding a novelmembrane protein responding to said RS38, accroding to screening a cDNAlibrary prepared from a synovial cell line derived from patients withrheumatoid arthritis (RA) by using the RS38 antibody, which has led tothe completion of the present invention.

That is, the present invention is directed to provide a novel membraneprotein polypeptide having pre-B cell growth-supporting ability, a geneencoding said polypeptide, a vector containing said gene, transformantstransformed by said vector and a method for producing a novel membraneprotein by using said gene.

Further, the present invention is directed to provide a monoclonalantibody recognizing a novel membrane protein having pre-B cellgrowth-supporting ability.

The present invention for accomplishing the above object consists of thefollowing (1)-(7).

(1) A novel membrane protein polypeptide containing an amino acidsequence shown in sequence No. 1 of the sequence table or a part of theamino acid sequence and being expressed on the synovial membrane ofpatients with rheumatoid arthritis.

(2) A DNA encoding a polypeptide containing an amino acid sequence shownin sequence No. 1 of the sequence table or a part of the amino acidsequence.

(3) The DNA according to the above (2), characterized by containing abase sequence which hybridizes the base sequence shown in sequence No. 2of the sequence table or a base sequence derived from said base sequencehaving at least one amino acid residue substituted, removed or addedpartially.

(4) A recombinant vector containing the DNA according to the above (2)or (3).

(5) A prokaryotic or eukaryotic host cell, characterized by beingtransformed with the recombinant vector according to the above (4).

(6) A method for producing the polypeptide containing an amino acidsequence shown in sequence No. 1 of the sequence table or a part of theamino acid sequence, characterized by culturing the host cell accordingto the above (5).

(7) A monoclonal antibody recognizing a polypeptide containing an aminoacid sequence shown in sequence No. 1 of the sequence table or a part ofthe amino acid sequence.

Subsequently, the present invention will be described in detail.

The monoclonal antibody of the present invention may be prepared in thefollowing manner essentially.

That is, the antibody of the present invention may be prepared by usinga synovial cell derived from patients with rheumatoid arthritis (RA)having pre-B cell growth-supporting ability as an antigen, immunizing itaccording to an ordinary immunization method, cell-fusing the immunizedcell according to an ordinary cell fusion method and cloning the fusedcell according to an ordinary cloning method.

More specifically, as a preferable method for producing the monoclonalantibody of the present invention may be exemplified a method comprisingusing the cell line SynSV6-14, derived from the synovial membrane ofpatients with rheumatoid arthritis (RA) and established as a culturecell, as the above-mentioned antigen, fusing the plasma cell(immunocyte) of a mammal immunized with said antigen with a myeloma cellof a mammal such as a mouse, cloning the obtained fused cell(hybridoma), selecting clones producing the antibody of the presentinvention recognizing SynSV6-14 of them, and culturing them to recoverthe objective antibody.

In the method for producing the above monoclonal antibody, mammals to beimmunized with the antigen are not particularly restricted; it ispreferable to select one taking compatibility with a myeloma cell to beused for cell fusion into consideration and generally, a mouse, a ratand a hamster are used.

Immunization is performed according to a general method, for example, byadministering cultured cells of the cell line SynSV6-14 derived from thesynovial membrane of patients with rheumatoid arthritis (RA) into theperitoneal cavity of a mammal according to injection. More specifically,it is preferable to dilute it with or suspend it in PBS or physiologicalsaline to a proper amount and administer it into an animal several timesevery 4-21 days, together with an ordinary adjuvant if required. Inaddition, an ordinary carrier (Schlepper) may be employed on the aboveadministration. As an immunocyte, a splenic cell obtained after thefinal administration of the above cell line is used preferably.

As a myeloma cell of a mammal as the other parent cell to be fused withthe above immunocyte may be preferably used known various cell linesincluding P3 (P3X63Ag8.653) [J. Immunol., 123:1548 (1978)], p3-U1[Current Topics in Micro-biology and Immunology, 81:1-7 (1978)], NS-1[Eur. J. Immumol., 6:511-519 (1976)], MPC-11 [Cell, 8:405-415 (1976)],SP2/0 [Nature, 276: 269-270 (1978)], FO [J. Immunol. Meth., 35:1-21(1980)], S194 [J. Exp. Med., 148:313-323 (1978)] and R210 [Nature,277:131-133 (1979)].

The cell fusion of the above immunocyte with a myeloma cell may beperformed essentially according to a known method, for example, a methodby Milstein et al. [Methods Enzymol., 73:3-46 (1981)].

More specifically, the above cell fusion may be performed, for example,in an ordinary nutrition medium in the presence of a fusion-acceleratingagent. Examples of the fusion-accelerating agent include polyethyleneglycol (PEG) and Sendai virus (HVJ), and moreover, auxiliary agents suchas dimethyl sulfoxide may be added properly if required in order toenhance the fusing effect. Regarding the ratios of immunocytes andmyeloma cells used, the former is preferably used in an amount 1-10times that of the latter. Examples of a medium used in the above cellfusion include an RPMI-1640 medium and an MEM medium suitable for theproliferation of the above myeloma cell line and other mediumsordinarily used for the culture of this kind of cell, and in addition,supplementary serum such as fetal calf serum (FCS) may be used together.

Cell fusion is performed by mixing prescribed amounts of the aboveimmunocytes and myeloma cells thoroughly in the above medium, adding aPEG solution preheated to about 37° C., for example, PEG with an averagemolecular weight of the order of 1,000-6,000, to the medium ordinarilyat a concentration of about 30-60% (W/V) and mixing them. Subsequently,by repeating the operations of adding proper mediums to themsuccessively and centrifuging the reaction mixture, and removing thesupernatants can be formed an objective hybridoma.

Said hybridoma is selected by culturing in an ordinary selective medium,for example, an HAT medium (medium containing hypoxanthine, aminopterinand thymidine). The culture in said HAT medium is continued for a timesufficient for cells other than objective hybridomas (non-fused cells)to die out, ordinarily for several days to several weeks. Subsequently,the screening and monocloning of the hybridomas producing the objectiveantibody are performed according to an ordinary limiting dilutionanalysis.

The thus prepared hybridomas producing the monoclonal antibody of thepresent invention may be subcultured in an ordinary medium and stored inliquid nitrogen for a long time.

In order to collect the monoclonal antibody of the present inventionfrom said hybridomas may be employed a method comprising culturing saidhybridomas according to an ordinary method and obtaining it from thesupernatants or a method comprising administering a hybridoma into aappropriate mammal to proliferate and obtaining it from its ascites. Theformer is suitable for obtaining an antibody with a high purity and thelatter is suitable for the mass production of the antibody.

Moreover, the antibody obtained according to the above method may bepurified to have a high purity employing an ordinary purification meanssuch as a salting out technique, gel filtration and affinitychromatography.

The thus prepared monoclonal antibody of the present invention makes itpossible to identify synovial cells of patients with rheumatoidarthritis (RA) expressing a novel membrane protein of an antigen with ahigh sensitivity and a high precision according to an ordinaryimmunological means such as radioimmunoassay (RIA), enzyme immunoassay(EIA) and immunofluorescence analysis.

The gene of the present invention is obtained by preparing mRNA from asynovial cell of patients with rheumatoid arthritis (RA) expressing amembrane protein having human pre-B cell growth-supporting ability, andthen converting it into a double-stranded cDNA according to a knownmethod. As a cell used for preparing the mRNA can be mentioned, forexample, a cell line SynSV6-14 used as an immune source of a hybridomaRS38, but it is not limited to the cell line and therefore any type ofcells expressing the membrane protein having human pre-B cellgrowth-supporting ability may be used. Incidentally, SynSV6-8 was usedin the present invention.

For the preparation of the total RNA for obtaining mRNA can be employeda method for obtaining the total RNA which consists of performing cesiumchloride density-gradient centrifugation after a guanidine thiocyanatetreatment [Chirgwin et al., Biochemistry, 18:5294 (1979)], a methodwhich consists of performing a surfactant treatment and a phenoltreatment in the presence of the ribonuclease inhibitor of a vanadiumcomplex [Berger & Birkenmeier, Biochemistry, 18:5143 (1979)], and otherknown methods.

The preparation of mRNA from the total RNA can be accomplished byrecovering poly(A)⁺RNA from the total RNA according to, for example,affinity column chromatography using an oligo (dT)-bound carrier, forexample, cephalose or cellulose, or a batch method. Besides, poly(A)⁺RNAcan be further purified according to sucrose density-gradientcentrifugation. In addition, there can be mentioned a method forobtaining poly(A)⁺RNA directly without preparing RNA or a convenientmethod using a commercially available kit.

In order to obtain a double-stranded cDNA from the thus obtained mRNA,for example, a DNA (cDNA) complementary to mRNA is synthesized by usingmRNA as a template, and using an oligo (dT) complementary to apoly-A-chain sited at the 3′ end as a primer, and then treating it withreverse transcriptase.

The double-stranded cDNA can be also obtained by degrading mRNAaccording to an alkaline treatment, subjecting the obtainedsingle-stranded cDNA as a template to a treatment with reversetranscriptase or DNA polymerase (e.g., Klenow fragment), and thentreating it with SI nuclease, or treating it directly with RNase and DNApolymerase [Maniatis et al., Molecular Cloning, Cold Spring HarborLaboratory (1982) and Gubler & Hoffman, Gene, 25:263 (1983)]. Nowadays,convenient kits have been on the market, and a double-stranded cDNA canbe obtained by using them.

The cDNA library can be obtained by inserting the thus obtained cDNAinto a proper vector, for example, an EK-type plasmid vector such aspBR322 and pSC101, and a phage vector such as λ gt10, and thentransforming Escherichia coli with said vector (e.g., X1776, HB101, DH1,DH5) or the like (refer, for example, to “Molecular Cloning” above).

On the other hand, host cells of other prokaryotes and eukaryotes can betransformed by using a suitable expression vector in which thedouble-stranded cDNA obtained according to the above-mentioned method isinserted.

The ligation of the double-stranded cDNA to the vector can be performedby adding a proper chemically-synthesized DNA adapter thereto, andsubjecting it with a vector DNA cleaved by means of a restriction enzymein advance to a treatment with T4 phage DNA ligase in the presence ofATP.

The expression vector of the present invention contains a replicativeorigin, a selective marker, a promoter located in the upstream region ofa gene to be expressed, an RNA splice site and a polyadenylated signal.

As a gene expression promoter in a mammal cell may be used viruspromoters such as retrovirus, polyoma virus, adenovirus and simian virus(SV) 40, and promoters derived from cells such as human polypeptidechain elongation factor 1α (HEF-1α). For example, in case of using apromoter of SV40, it can be performed easily according to a method ofMulligan et al. [Nature, 277:108 (1979)].

As a replicative origin can be used those derived from SV40 polyomavirus, adenovirus and bovine papilloma virus (BPV), and as a selectivemarker can be used a phosphotransferase APH (3′) II or I (neo) gene, athymidine kinase (TK) gene, an Escherichia coli xanthine-guaninephosphoribosyl transferase (Ecogpt) gene and a dihydrofolate reductase(DHFR) gene.

In order to express the desired gene using a prokaryotic cell as a hostcell, the host cell is transformed with a replicon derived from speciescapable of being fitted for hosts, namely, a plasmid vector containing areplicative origin and a regulation sequence. A vector which has amarker gene capable of imparting the selectivity of a phenotype totransformed cells is preferable. For example, in case of usingEscherichia coli as a host cell, it can be transformed using pBR322, avector originated from the host cell [Boliver et al., Gene, 2:95(1975)]. The pBR322 contains an ampicillin resistant gene and atetracycline resistant gene, and therefore transformants can beidentified by utilizing either of these resistant properties.

As a promoter needed for the gene expression of a prokaryotic host cellcan be mentioned a promoter of a β-lactamase gene [Chang et al., Nature,275:615 (1978)], a lactose promoter [Goeddle et al., Nature, 281:544(1979)], a tryptophan promoter [Goeddle et al., Nucleic Acid Res.,8:4057 (1980)], a tac promoter and the like preferably; however, it isnot limited to them.

As a prokaryotic host cell of hosts to be used in the expression systemof the present invention can be mentioned Escherichia coli, Basillussubtilis, Bacillus thermophilus and the like preferably; however, it isnot limited to them.

In addition, as an eukaryotic host cell can be mentioned eukaryoticmicroorganisms such as Saccharomyces cerevisiae, and cells derived frommammals such as a COS cell, a Chinese hamster ovary (CHO) cell, a C127cell, a 3T3 cell, a Hela cell, a BHK cell, a namalwa cell and a humanfetal renal cell (293 cell) preferably; however, it is not limited tothem.

Incidentally, the culture of the transformants of the present inventionmay be performed by selecting culture conditions suitable for host cellsappropriately.

The isolation of a cDNA encoding a membrane protein having pre-B cellgrowth-supporting ability of the present invention can be performed, forexample, by using pre-B cell growth-supporting ability as an index oraccording to a method such as direct expression cloning using anantibody.

The measurement of pre-B cell growth-supporting ability can be performedby using a murine pre-B cell line DW34 [Eur. J. Immunol., 18:1767(1988)]. That is, a cell expressing the membrane protein having pre-Bgrowth-supporting ability is cultured until it becomes subconfluent on24-well plates (preferable density being about 50%) and a proper amountof radiation is irradiated thereupon, DW34 of 1 to 2×10³ per well isadded thereto, and cultured in the RPMI-1640 medium containing 10% FCSunder the condition of 5% CO₂ at 37° C. for about 4 to 6 days. Thedegree of the enhancement of the growth-supporting ability can be foundby examining the number of viable cells of DW34 in each well accordingto trypan blue dye exclusion.

In the present invention, the desired gene could be cloned by repeatingthe steps, which consist of selecting a transformant expressing amembrane protein according to flow cytometry by means of an FACScanusing a monoclonal antibody RS38 recognizing the novel membrane proteinon the synovial cell of patients with rheumatoid arthritis (RA),preparing a transformant again by sorting the plasmid DNA used for thepreparation of the transformant, and then screening the transformantaccording to flow cytometry.

Specifically, a transduced transformant (293T cell) was cultured on wellplates and removed from the plates with PBS containing 0.02% EDTA, andafter the cell was washed with an FACS buffer solution composed of PBScontaining 2% FCS and 0.02% NaN₃, it was reacted with RS38 as a primaryantibody. Subsequently, after the unreacted primary antibody was removedby washing it with an FACS buffer solution, it was further reacted witha secondary antibody, an FITC-labeled antibody (FITC-labeled anti-mousegoat Ig antibody), dead cells were stained with propidium iodide, andviable cells were analyzed by an FACScan to select transformantsresponding strongly to RS38.

Further, the complete length of cDNA (pRS38-BOS) encoding a membraneprotein polypeptide having novel pre-B cell growth-supporting abilityshown in sequence No. 2 of the sequence table could be obtained byrepeating the steps, which consist of treating Escherichia coli (DH5)containing the cDNA used for the preparation of transformants respondingto the antibody with alkali to select a group of plasmids containing thedesired gene, subdividing the group of plasmids into some groups ofplasmids, transducing them into 293T cells again, and then selectingtransformants according to FACScan analysis using the above-mentionedmonoclonal antibody RS38.

Incidentally, the Escherichia coli DH5α strain containing pRS38-pUC19with the cDNA inserted into the XbaI cleavage sites of a pUC19 vectorwas deposited at National Institute of Bioscience & Human Technology,Agency of Industrial Science and Technology in Japan, which is aninternational depositary authority according to Budapest Treaty on theinternational recognition of the deposit of microorganisms for thepurpose of patent procedure, on Oct. 5, 1993, under the name ofEscherichia Coli DH5α (pRS38-pUC19) with accession No. FERM BP-4434.

Generally, the genes of eukaryotes are thought to show polymorphism asknown according to human interferon genes [e.g., Nishi et al., J.Biochem., 97: 153 (1985)], and in some cases at least one amino acid issubstituted according to this polymorphism, and in other cases aminoacids do not change at all though there are changes in the DNA sequence.

Further, it is probable that some polypeptides having at least one moreor less amino acid than the amino acid sequence shown in sequence No. 1of the sequence table, or some polypeptides substituted with at leastone amino acid may also have the same function as that of the novelmembrane protein of the present invention (pre-B cell growth-supportingability). Actually, for example, it has been already known that thepolypeptide obtained from a human interleukin-2 (IL-2) gene, in which aDNA sequence corresponding to cysteine is converted to a sequencecorresponding to serine, also holds an IL-2 activity [Wang et al.,Science, 224:1431 (1984)].

Moreover, a known protein gene and a gene shown in sequence No. 2 of thesequence table can be ligated by means of a proper restriction enzyme oradapter to yield a polypeptide bound to the known protein. As the knownprotein gene can be mentioned immunoglobulin, and it may be bound to aFc portion thereof using the gene shown in sequence No. 2 of thesequence table instead of the variable region site thereof [(Zettlmeisslet al., DNA AND CELL BIOLOGY, 9:347-353 (1990)].

Furthermore, in case of expressing a polypeptide in eukaryotic cells,glycosylation occurs in many cases, and the glycosylation can beregulated according to the conversion of at least one amino acid; inthis case, too, it may have the same function as that of the novelmembrane protein polypeptide of the present invention. Therefore, eventhe genes in which the site encoding the membrane protein polypeptide ofthe present invention are modified artificially according to variousmethods as above and polypeptides can be included in the presentinvention so far as the polypeptides obtained from the genes have thesame function as that of the membrane protein polypeptide of the presentinvention.

Moreover, it goes without saying that genes to be hybridized with genesshown in sequence No. 2 of the sequence table and polypeptides are alsoincluded in the present invention so far as the polypeptides expressedfrom the genes have the same function as that of the membrane proteinpolypeptide of the present invention (pre-B cell growth-supportingability). In this case, hybridization may be carried out according toemploying ordinary hybridization conditions (for example, refer to theabove-mentioned “Molecular Cloning”).

The desired homogeneous and purified soluble membrane proteinpolypeptide having pre-B cell growth-supporting ability can be obtainedby culturing a transformant transformed with a gene encoding thepolypeptide, solubilizing the yielded polypeptide with a properdetergent, subjecting the resultant polypeptide to separation andpurification. Preferable examples of the detergent include Nonidet P-40(NP-40), Sodium Dodecyl Sulphate (SDS), Triton X-100, Tween 20 and thelike.

In addition, soluble membrane proteins can be also prepared according togene engineering. Namely, as shown in FIG. 3, since RS38 is guessed tobe a cell membrane through-type protein having a cell membrane throughdomain and an intracellular domain at the side of the N terminal,soluble RS38 with the 49th Asn of sequence No. 1 of the sequence tableas the N end can be prepared by employing a PCR-mutagenesis method [M.Kamman et al., Nucl. Acids Res., 15:5404 (1989)]. In this case, as asignal sequence may be used known ones and examples thereof include thesignal sequence of Bst-1 (Japanese Patent Application No. 5-141178/1993)and that of G-CSF (Japanese Patent Publication No. 2-5395/1990).

As a means of separation and purification of the membrane proteinpolypeptide, a method to be used in the case of ordinary protein can beemployed; for example, the membrane protein polypeptide of the presentinvention can be separated and purified properly by selecting andcombining various types of chromatograpy such as affinity chromatographyusing the above-mentioned monoclonal antibody, ultrafiltration, saltingout, dialysis and the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results (photograph according to agarose gelelectrophoresis) of analyzing a gene obtained in Examples of the presentinvention according to northern blotting analysis.

FIG. 2 shows the growth ability of the mouse pre-B cell line DW34 of amembrane protein obtained in Examples of the present invention.

FIG. 3 shows the results of analyzing the hydrophobic region and thehydrophilic region of a gene obtained in Examples of the presentinvention by means of a DNA analysis software Gene Works.

The present invention will be described in detail according toReferential Examples and Examples hereinafter, although the presentinvention is not limited to these Examples.

REFERENTIAL EXAMPLE 1

Establishment of a Stromal Cell Line Derived from Healthy Donors

Stromal cells derived from healthy donors were electroporated with apAct-SVT plasmid containing an SV40 large T antigen cDNA and a chickβ-actin promoter [BBRC, 186:129-134 (1992)] by means of a Gene Pulser(manufactured by BioLad). Namely, 0.8 ml of an aliqout of the stromalcells of 1×10⁷ cells/ml derived from healthy donors in PBS were mixedwith 10 μg of the plasmid, and the mixture was incubated on ice for 10minutes, subjected to electroporation under the conditions of 250 V andat an electrostatic capacity of 250 μF, further incubated on ice for 10minutes, suspended in the RPMI-1640 medium (manufactured by GIBCO)containing 10% FCS (manufactured by Bioproducts), and cultured in a10-centimeter culture dish. The culture medium was changed every threedays, and colonies of well-grown adhesive cells were harvested about 2weeks later with a small piece of filter paper impregnated with trypsinto obtain a stromal cell line (NFSV1-1) derived from the bone marrow ofhealthy donors [J. Immunol., 149:4088 (1992)].

REFERENTIAL EXAMPLE 2

Establishment of Synovial Cell Lines Derived from Patients withRheumatoid Arthritis (RA)

Synovial cells derived from patients with rheumatoid arthritis (RA) wereelectroporated with a pAct-SVT plasmid containing an SV40 large Tantigen cDNA and a chick β-actin promoter [BBRC, 186:129-134 (1992)] bymeans of a Gene Pulser (manufactured by BioLad). Namely, 0.8 ml of analiqout of the synovial cells of 1×10⁷ cells/ml derived from patientswith RA in PBS were mixed with 10 μg of the plasmid, and the mixture wasincubated on ice for 10 minutes, subjected to electroporation under theconditions of 250 V and at an electrostatic capacity of 250 μF, furtherincubated on ice for 10 minutes, suspended in the RPMI-1640 medium(manufactured by GIBCO) containing 10% FCS (manufactured byBioproducts), and cultured in a 10-centimeter culture dish. The culturemedium was changed every three days, and colonies of well-grown adhesivecells were harvested about 2 weeks later with a small piece of filterpaper impregnated with trypsin to obtain synovial cell lines (SynSV6-8and SynSV6-14) derived from patients with rheumatoid arthritis (RA).

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiment of the present invention will be described in detailhereinafter.

EXAMPLE 1

Preparation of Monoclonal Antibodies

1) Antigen and Immunization

The synovial cell line SynSV6-14 derived from patients with rheumatoidarthritis (RA) having pre-B cell growth-supporting ability obtained inthe above Referential Example 2 was used as an antigen for immunization.Using the RPMI-1640 medium (manufactured by GIBCO) containing 10% fetalcalf serum (FCS, manufactured by Bioproducts) and 50 μM2-mercaptoethanol as a medium, the cell line was subcultured in anincubator containing 5% CO₂, at 37° C.

The cells were treated with 0.02% EDTA and PBS, and recovered from aculture flask of the incubator according to pipetting. The cells weresuspended into the RPMI medium at a rate of about 1×10⁷ cells/ml, andimmunized to a BALB/C mouse (4-week old, female, manufactured by S. L.C. of Japan). In the initial immunization, about 1×10⁷/ml cells wereinjected into the peritoneal cavity of the mouse, and 2 to 3 weekslater, 1×10⁷/ml cells were injected as additional immunization. Further,at intervals of 2 to 3 weeks, 1×10⁷/ml cells were injected 2 to 3 timesas additional immunization, and 3 days after the final immunization, themouse was sacrificed and the spleen was obtained for fusion.

2) Cell Fusion

After the spleen extirpated from one mouse was cut to pieces, isolatedspleen cells were centrifuged, suspended into RPMI-1640 medium(manufactured by GIBCO), and washed sufficiently. On the other hand,1×10⁷ cells obtained by culturing a mouse-myeloma cell line P3X63Ag8.653[J. Immunol., 123:1548 (1979)] in the DMEM medium (manufactured byGIBCO) containing 10% fetal calf serum (FCS, manufactured by FILTRON)were washed in the above DMEM medium similarly, and were introduced intoa centrifugal tube together with 1×10⁸ of said spleen cells and mixed,and then were subjected to cell-fusion with polyethylene glycol 1500(manufactured by Boehringer) according to ordinary procedure [Clin. Exp.Immunol., 42:458-462 (1980)].

The obtained fused cells were introduced into 96-well plates in the DMEMmedium containing 10% FCS, and cultured in an incubator containing 5%CO₂, at 37° C. From the following day, the medium was replaced with theHAT selective medium (complete RPMI-1640 medium containing 1.0×10⁻⁴Mhypoxanthine, 4.0×10⁻⁷ M aminopterin and 1.6×10⁻⁵M thymidine having 10%FCS and 50 μM 2-mercaptoethanol added thereto) slowly, and the culturewas continued. After the culture was initiated, half of the supernatantwas replaced with a new HAT medium 2 times per week, and the culture wascontinued to maintain proliferation.

The thus obtained fused cells were cloned according to limiting dilutionanalysis.

That is, using the antibodies in the culture supernatant obtained byculturing the above fused cells, respondency with the antigen wasexamined, and clones having strong respondency with the antigen alonewere obtained according to ordinary procedure employing limitingdilution analysis.

So as to perform the formation of clones, the above hybridoma and thespleen cells of a BALB/C mouse were prepared in prescribed amounts,inoculated onto 96-well plates at a rate of 1 to 10 hybridoma(s) perwell, and cultured in an incubator containing 5% CO₂, at 37° C. Theoperation of cloning hybridomas grown was repeated in the same manneraccording to ordinary limiting dilution analysis until they became asingle clone theoretically. Clones yielding the desired antibody werescreened using the above antigen.

3) Screening

The screening of fused cells (hybridomas) was performed according toindirect fluorescent antibody technique, flow cytometry analysis bymeans of a flow cytometer.

The screening of clones yielding the objective antibody was performedusing a) SynSV6-14 (antigen for immunization) and b) the bone marrowstromal cell line NFSV1-1 derived from healthy donors as target cells.Namely, after immunizing the synovial cell line (SynSV6-14) derived frompatients with rheumatoid arthritis (RA) having pre-B cellgrowth-supporting ability to a BALB/C mouse, the screening of monoclonalantibodies responding to SynSV6-14 but not responding to the bone marrowstromal cell line (NFSV1-1) derived from healthy donors having no pre-Bcell growth-supporting ability was performed as follows. The firstscreening was performed using SynSV6-14, a cell to be subjected toreaction, as an antigen for immunization. First of all, culturesupernatants responding to SynSV6-14 were selected with a view toselecting fused cell clones responding to said SynSV6-14, and then aprimary screening was performed.

Namely, cells suspended in a reaction buffer (PBS containing 2% FCS and0.02% NaN₃) were suspended into 20μ1 of a hybridoma culture supernatant(about 5×10⁵/20μ1), and reacted at 4° C. for 20 minutes.

They were washed with the above buffer twice, and the FITC-labeledanti-mouse Ig goat antibody (manufactured by Cappel) was added thereto,and the mixture was incubated for 20 minutes. After the reaction productwas washed three times, it was analyzed by means of a flow cytometer(FACScan, manufactured by Becton Dickinson).

Subsequently, the bone marrow stromal cell line NFSV1-1 derived fromhealthy donors was used as a cell to be subjected to reaction andanalyzed by means of a flow cytometer as above. A hybridoma yieldingantibodies responding more strongly to SynSV6-14 was obtained accordingto it.

Thus, a hybridoma (RS38) yielding antibodies responding to SynSV6-14 butnot responding to the bone marrow stromal cell line NFSV1-1 derived fromhealthy donors was isolated. The antibodies yielded by the hybridomawere IgM, κ-type.

Incidentally, the hybridoma yielding the above monoclonal antibody RS38is a novel fused cell prepared from a BALB/C mouse spleen cell andmouse-myeloma P3X63Ag8.653 as parent, cells and was deposited atNational Institute of Bioscience & Human Technology, Agency ofIndustrial Science and Technology, AIST Tsukuba Central 6, 1-1, Higashi1-chome, Tsukuba-shi, Ibaraki-Ken 305-8566, Japan, which is aninternational depositary authority according to Budapest Treaty on theinternational recognition of the deposit of microorganisms for thepurpose of patent procedure, on Oct. 5, 1993, under the name ofMouse-Mouse hybridoma RS38 with accession No. of FERM BP-4433.

4) Purification of Antibodies

The fused cells prepared in the above 2) were cultured according toordinary procedure, and the antibodies yielded in the culturesupernatant were purified according to ordinary procedure.

That is, hybridomas were collected from the wells with the highestantibody titer to the above antigen, and one well in which the growth ofcells could be recognized was taken out, and the obtained culture cellswere expanded into a tissue culture flask under the conditions of 5% CO₂at 37° C. and were grown. The obtained cells were injected into theperitoneal cavity of a BALB/C mouse (6-week old, female, manufactured byS. L. C. of Japan) with pristan dosed. And 10 to 14 days after, theascites was collected, salted out with 50% ammonium sulfate, dialyzedwith PBS and purified with a QAE column. The antibodies were furthersalted out and dialyzed sufficiently to obtain a purified product ofabout 6 mg/ml.

EXAMPLE 2

Properties of the Monoclonal Antibodies

1) Distribution of Cell Lines

The distribution of the RS38 antigen in various cell lines (various celllines shown in Table 1) was analyzed by means of a FACScan. That is,each of various cell lines was suspended in an FRCS buffer solutioncomprising PBS containing 2% FCS and 0.02% NaN₃, in the presence of 10μg/ml of the mouse monoclonal antibody RS38 obtained as a primaryantibody in Example 1, and incubated on ice for 20 minutes. After washedwith an FACS buffer solution twice, the resultant product was furtherincubated on ice for 15 minutes using an FITC-labeled anti-mouse Ig goatantibody (manufactured by Cappel) as a secondary antibody. Propidiumiodide (PI) was added therein so that the final concentration of itbecame 1 μg/ml, and the mixture was further incubated on ice for 5minutes. After the resultant product was washed with the FACS buffersolution three times, the cells were analyzed with light scatteringmeasurement and viable cells alone were subjected to analysis by meansof an FACScan (manufactured by Becton Dickinson). As controls were usedmouse monoclonal antibodies SG2 and RF3 (Japanese Patent Application No.5-141178/1993). The results are shown in Table 1. As is apparent fromTable 1, it was revealed that the distribution of the RS38 is differentfrom that of SG2 and RF3. TABLE 1 Results of FACS analysis on Cell LinesOrigen Cell Line SG2/RF3 RS38 T cell line MOLT-4 − +/− JURKAT + − B cellline Raji − − Paudi − − CL4 − ++ Ramos − − Myeloid U937 + + K562 − +HL60 − +/− MO7 − + Miscellaneous HepG2 − +/− T24 − +/− SKWG-4(P122) −+/− Hela − +/−

EXAMPLE 3

1. Preparation of a cDNA Library

1) Preparation of Poly (A)⁺RNA

The preparation of poly (A)⁺RNA from the synovial cell line SynSV6-8derived from patients with rheumatoid arthritis (RA) was performed usinga Fast Track™ mRNA isolation kit version 3.2 (manufactured byInvitrogen). That is, SynSV6-8 cells for twenty 10-centimeter culturedishes were homogenized, and then the total RNA was prepared accordingto the procedure attached to the kit. Further, the poly (A)⁺RNA waspurified by means of oligo d(T) cellulose attached to the kit accordingto the procedure attached to the kit.

2) Construction of the cDNA Library

A double-stranded cDNA was synthesized using the above poly (A)⁺RNA of 5μg as a material according to the procedure attached to a cDNA synthesiskit, Time Saver™ cDNA synthesis kit (manufactured by Pharmacia), and aBstXI adapter (manufactured by Invitrogen) was ligated thereto by meansof a DNA ligation kit (manufactured by Takara Shuzo) according to theprocedure attached to the kit. The removal of the free BstXI adapter wasperformed by means of the Size Sep 400 Spin Column attached to the kitaccording to the procedure attached to the kit to obtain about 100 μl ofan adapter-ligated double-stranded cDNA.

And then, of about 100 μl of the thus prepared adapter-ligateddouble-stranded cDNA, 2 μl were used in one ligation reaction, and acDNA library was constructed by ligating it with a pEF-BOS vector [Nuc.Acid Res., 18:5322 (1990)] treated in advance with a restriction enzymeBstXI and alkali phosphatase (manufactured by Takara Shuzo) by means ofa cDNA ligation kit (manufactured by Takara Shuzo). The constructed cDNAlibrary was transformed into the Escherichia coli cell line DH5(manufactured by Toyobo) and was presumed to be an independent clonewith the total size of about 2×10⁵. Fifty pools, each pool comprising2,000 to 4,000 clones of transduced Escherichia coli, were prepared andthen used in the following tests.

2. Cloning According to Direct Expression Method

1) Transfection into 293T Cells

The amplification of a cDNA was performed by culturing the above pooledEscherichia coli in the LB medium containing 50 μg/ml of ampicillin[Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold SpringHarbor Laboratory Press (1989)], and a plasmid DNA was recovered fromthe Escherichia coli according to an alkaline method [Molecular Cloning:A Laboratory Manual, Sambrook et al., Cold Spring Harbor LaboratoryPress (1989)]. The degree of the purification of the obtained plasmidDNA was enhanced by repeating ultra-centrifugation according to cesiumchloride/ethidium bromide density-gradient centrifugation, and thepurified plasmid DNA was transfected to a 293T cell [cell line preparedby transfecting an SV40 large T antigen cDNA into a 293 cell(Transformed primary embryonal kidney, human ATCC CRL 1573)] accordingto a calcium phosphate method.

Namely, 2 μg of the purified plasmid DNA was dissolved into 100 μl of abuffer solution containing 1 mM of Tris-HCl and 0.1 mM of EDTA, andafter 14 μl of 2M CaCl₂ were added thereto, the resultant mixture wasmixed with a buffer solution composed of 50 mM of HEPES (pH: 7.1), 280mM of NaCl and 1.5 mM of sodium phosphate slowly, and then the obtainedmixture was incubated at room temperature for 30 minutes and added tothe 293T cells in 24-well plates. The 293T cells were cultured in theDMEM (manufactured by GIBCO) medium containing 10% fetal calf serum(FCS, manufactured by Bioproducts) under the conditions of 37° C. and 5%CO₂ for 2 days.

2) Analysis According to FACScan

The transduced 293T cells were removed from the 24-well plates in PBScontaining 0.02% EDTA, and washed with an FACS buffer solutioncomprising PBS containing 2% FCS and 0.02% NaN₃ twice, and thensuspended in 20 μl of the FACS buffer solution in the presence of 10μg/ml of the above-mentioned monoclonal antibody RS38 as a primaryantibody and incubated on ice for 20 minutes. After they were washedwith the FACS buffer solution twice, they were further incubated on icefor 15 minutes, using an FITC-labeled anti-mouse Ig goat antibody(manufactured by Cappel) as a secondary antibody. Propidium iodide (PI)was added therein so that the final concentration of it became 1 μg/ml,and the mixture was further incubated on ice for 5 minutes, washed withthe FACS buffer solution three times, and the cells were analyzed withlight scattering measurement and viable cells alone were subjected toanalysis by means of an FACScan (manufactured by Becton Dickinson).

3) Cloning of the cDNA Library

The plasmid DNAs recovered from Escherichia coli of 2,000 to 4,000clones as one pool according to an alkaline method were transfected to293T cells according to the above method, and the transfected cells weresubjected to screening according to the above FACS analysis. A peakstrongly stained with the mouse monoclonal antibody RS38 was recognizedin the 25th pool of the 293T cells. The plasmid DNA was transduced intoEscherichia coli DH5 (manufactured by GIBCO BRL) again, and it wasinoculated onto an LB agar plate containing 50 μg/ml of ampicillin.

About 2,000 clones forming colonies were inoculated one by one onto anagar plate with its bottom divided into a net-like state so that theposition of a clone inoculated was recognized at a rate of 100 clonesper plate, and two series of each plate were prepared. 20 pools, eachpool comprising 100 clones, were prepared in the same way, andEscherichia coli was cultured in the LB medium containing 50 μg/ml ofampicillin. After plasmid DNAs were recovered according to an alkalinemethod, they were transfected to 293T cells according to a calciumphosphate method, and the transfected cells were subjected to FACScananalysis in the same manner as above. As a result of the FACScananalysis, 100 clones of Escherichia coli were isolated one by one fromone pool recognized to be positive, and each clone was cultured, andthen plasmid DNAs were recovered according to an alkaline method. Eachplasmid DNA was transfected to 293T cells according to a calciumphosphate method, and FACScan analysis was performed in the same manneras above to obtain a single positive clone, which was designated aspRS38-BOS.

The clone was subjected to a sequence reaction using an Auto Readsequencing kit (manufactured by Pharmacia) and an Auto Cycle sequencingkit (manufactured by Pharmacia) according to the procedure attached tothe kits, and the determination of its DNA sequence was performed bymeans of an A. L. F.™ DNA sequenator (manufactured by Pharmacia). As aresult, it was a gene with the full length of 996 bp (sequence No. 2 ofthe sequence table) to be presumed to encode a sequence of the 180 aminoacid residues from the longest open reading frame, and as a result ofperforming homology retrieval according to data bases SWISS PLOT andNBRL using a gene analysis software Genetex, it was recognized to be anovel gene.

4) Investigation of Expression According to Northern Blotting Analysis

Preparation of poly (A)⁺RNA from the synovial cell line SynSV6-14derived from patients with RA, the bone marrow stromal cell line RASV5-5derived from patients with RA and the bone marrow stromal cell lineNFSV1-1 derived from healthy donors was performed by using a Fast Track™mRNA isolation kit version 3.2 (manufactured by Invitrogen) to performnorthern blotting analysis (Molecular Cloning; A Laboratory Manual,Sambrook et al., Cold Spring Habor Laboratory Press, 1989). That is,cell lines cultured in ten 10-centimeter culture dishes were recoveredand homogenized, and then the total RNA was prepared according to theprocedure attached to the kit. Further, the poly (A)⁺RNA was purified bymeans of oligo d(T) cellulose attached to the kit according to theprocedure attached to the kit.

Labelling a probe was performed by using a Multiprime DNA labellingsystem (manufactured by Amersham). That is, a 315 bp fragment wasprepared by digesting an insert believed to encode RS38 inserted intothe BstXI site of PEF-BOS with a restriction enzyme HindIII and then alabelled probe was prepared according to the procedure attached to thekit. The poly (A)⁺RNA prepared from SynSV6-14, RASV5-5 and NFSV1-1, 3 μgper lane, was subjected to agarose gel electrophoresis, and thenblotting was performed according to a capillary method by using a GeneScreen Plus™ (manufactured by Du Pont) as a hybridization transfermembrane. Hybridization was performed at 65° C. overnight by using aGilbert & Church buffer comprising 0.5 M NaPO₄, 1 mM EDTA, 7% SDS and 1%BSA, pH 7.0. After the completion of hybridization, the membrane waswashed with 2×SSC at room temperature four times and further with0.1×SSC and 0.1% SDS at 55° C. twice, and then placed face to face withan X-ray film to perform autoradiography overnight. As a result, anapparent band of about 1.0 kb was detected on SynSV6-14 as shown in FIG.1.

3. Expression by a BALB3T3 Cell

The novel molecule was transfected into a BALB3T3 cell and theexpression in mammalian cells was examined.

Namely, 20 μg of pRS38-BOS and 2 μg of pSv2neo which has aneomycin-resistant gene [P. J. Souethem and P. Berg, J. Mol. Appl.Genet., 1:327 (1982)] were added into 0.8 ml of aliquot of 1×10⁷cells/ml, and incubated on ice for 10 minutes, and then subjected totransfection by means of a Gene Pulser (manufactured by BioLad) underthe conditions of 250 V and an electrostatic volume of 250 μF toaccomplish transduction simultaneously.

Further, after it was incubated on ice for 10 minutes, the cell wassuspended in the DMEM medium (manufactured by GIBCO) containing 2 mg/mlof G418 and 10% FCS (manufactured by Bioproducts) and cultured in24-well plates. The replacement of a culture medium was performed everythree days, and about 2 weeks later, transformed cell lines BALB3T338-1, 38-3 and 38-9 responding to the above mouse monoclonal antibodyRS38 were obtained from the well forming a single colony of well-grownadhesion cells having neomycin resistance. In addition, as a controlcell was obtained a transformed cell line BALB3T3 38-4 not responding toRS38 but having neomycin resistance.

4. Biological Properties of the Novel Molecule

1) Growth-Supporting of Mouse Pre-B Cell Lines

The biological properties of the novel molecule were analyzed using amouse pre-B cell line DW34 growing dependent on stromal cells with thenumber of grown cells as an index according to the following method.

First of all, transduced cell lines BALB3T3 38-1, 38-3 and 38-9 andcontrol cell lines BALB3T3 and BALB3T3 38-4 were cultured in 24-wellplates until they became subconfluent, radiation of 30 Gy was irradiatedthereupon, DW34 of 2×10³ per well was added therein, and the resultantproduct was cultured in the RPMI-1640 (manufactured by GIBCO) mediumcontaining 10% FCS (manufactured by Bioproducts) under the conditions of37° C. and 5% CO₂ for 4 days. The number of viable cells of DW34 in eachwell was counted with trypan blue dye exclusion to analyze agrowth-supporting ability. As a result, the growth of DW34 was enhancedin the transduced cell lines BALB3T3 38-1, 38-3 and 38-9 in comparisonwith control cell lines BALB3T3 and BALB3T3 38-4 as shown in FIG. 2.

5. Physicochemical Properties of the Novel Molecule

1) Analysis of the N Terminal

The analysis of the hydrophobic region and the hydrophilic region of thegene obtained as above was performed by using a DNA analysis softwareGene Works. The results of the analysis are shown in FIG. 3. As aresult, the region of 28 amino acid residues from the 21st Lys to the48th Ala shown in sequence No. 1 of the sequence table had the highesthydrophobic properties, and therefore it was presumed to be type of aprotein to pass through the cell membrane, having a domain passingthrough cell membrane and an intracellular domain at the side of the Nterminal of mature protein.

INDUSTRIAL APPLICABILITY

As described in detail above, the present invention relates to a geneencoding a novel membrane protein polypeptide having pre-B cellgrowth-supporting ability, a vector containing said gene, transformantstransformed by said vector, and a method for producing a novel membraneprotein having pre-B cell growth-supporting ability using said gene, andtherefore the gene of the present invention is capable of encoding themembrane protein on the synovial cell derived from patients withrheumatoid arthritis (RA).

The homogeneous and purified novel membrane protein polypeptide can beproduced in large quantities by inserting the gene of the presentinvention into a suitable vector and then transforming ordinary hostcells, and in addition, monoclonal antibodies recognizing said membraneprotein polypeptide can be produced by using the synovial cell derivedfrom patients with rheumatoid arthritis (RA) as an antigen forimmunization. Thus, according to the present invention, it becomespossible to identify rheumatoid arthritis (RA) and also prepare reagentsfor the clinical diagnosis thereof.

1-7. (canceled)
 8. A method for identifying a synovial cell comprising:contacting a synovial cell with an antibody for a time and underconditions suitable for binding of the antibody to the synovial cell,and measuring binding, wherein said antibody has a positive reactivityagainst cell lines U937 and K562 and recognizes a polypeptide consistingof the amino acid sequence shown in SEQ ID NO: 1, said polypeptidehaving pre-B cell growth-supporting ability.
 9. The method of claim 8,wherein said synovial cell is from a subject having rheumatoidarthritis.
 10. A method for identifying a subject having rheumatoidarthritis comprising: contacting a synovial cell from a subjectsuspected of having rheumatoid arthritis with an antibody for a time andunder conditions suitable for binding, and determining binding of saidantibody to said synovial cell, wherein the occurrence of, or the amountof, binding identifies rheumatoid arthritis in said subject, whereinsaid antibody has a positive reactivity against cell lines U937 and K562and recognizes a polypeptide consisting of the amino acid sequence shownin SEQ ID NO: 1, said polypeptide having pre-B cell growth-supportingability.